Biology subjects

Sasazawa, M.

Publications and source records attributed to Sasazawa, M..

3 recordsLinked to original sources

bioRxiv2025

Bacterial Ribonucleoprotein bodies maintain an acidic pH environment as a mechanism of enzyme regulation

Phase separated biomolecular condensates create subcellular niches, yet their role in client regulation remains unclear. Here, we demonstrate that Bacterial Ribonucleoprotein bodies (BR-bodies) are acidic. Using ratiometric fluorescent probes in vivo, we find BR-bodies exhibit a dense-phase pH of [~]5.1, significantly lower than the near-neutral cytoplasm. Single-molecule localization microscopy and fluorescence lifetime imaging reveals that Caulobacter crescentus BR-bodies have spatially variable and acidic nanoscale RNase E clusters. These results question the notion of homogeneous condensates, suggesting that BR-bodies exhibit structural and biochemical diversity, which may facilitate RNA processing under stress. In vitro, pH gradients observed with C-SNARF-4F and RNase E CTD-pHluorin2 deteriorate with increasing buffer concentrations. Notably, the acidic microenvironment within BR-bodies enhances PNPase activity, highlighting the significance of condensate pH regulation. These findings suggest that pH modulation is intrinsic to condensates, directly influencing biochemical reactions and offering a new strategy for designing pH-sensitive drugs to target enzymes within condensates.

microbiology↗

bioRxiv2025

Multivalency controls the growth and dynamics of a biomolecular condensate

Biomolecular condensates are essential for cellular organization and function, yet understanding how chemical and physical factors govern their formation and dynamics has been limited by a lack of non-invasive measurement techniques. Conventional microscopy methods often rely on fluorescent labeling and sub-strate immobilization, which can perturb the intrinsic properties of condensates. To overcome these challenges, we apply label-free, contact-free holographic video microscopy to study the behavior of a condensate-forming protein in vitro. This technique enables rapid, high-throughput, and precise measurements of individual condensate diameters and refractive indexes, providing unprecedented insight into size distributions and dense-phase macromolecular concentrations over time. Using this method, we investigate the kinetics of droplet growth, aging, and equilibrium dynamics in the model condensate-forming protein PopZ. By systematically varying the concentration and valence of cations, we uncover how multivalent ions influence condensate organization and dynamics, a hypothesis we further test using super-resolution microscopy. Our findings reveal that PopZ droplet growth deviates from classical models such as Smoluchowski coalescence and Ostwald ripening. Instead, we show that condensate growth is consistent with gelation at the critical overlap concentration. Holographic microscopy offers significant advantages over traditional techniques, such as differential interference contrast (DIC) microscopy, delivering reproducible measurements and capturing condensate dynamics with unparalleled precision. This work highlights the power of holographic microscopy to probe the material properties and mechanistic underpinnings of biomolecular condensates, paving the way for deeper insights into their roles in synthetic systems.

biophysics↗

bioRxiv2024

PEPCy: Photostable fluoromodules for live cell, super-resolution microscopy of surface proteins

We report the evolution and application of two genetically encoded tags that bind the cyanine dyes Cy3 or Cy5 with high specificity and selectivity, in addition to enhancing their photostability. These tags, which we call PEPCy, can be used to target membrane proteins such as G-protein coupled receptors. Due to their orthogonality and high binding-affinity for cognate cyanine dyes, the PEPCy tags can be used for wash-free labeling of cell surface receptors to observe their dynamics at a single molecule level. Together with self-labeling tags, these photostability enhancing proteins against cyanine dyes present a novel, complementary and powerful approach to explore protein dynamics with high spatiotemporal resolution.

biophysics↗